Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.
ADAMTS13 (A Disintegrin And Metalloprotease with ThromboSpondin type1 repeats-13) cleaves ultra large (UL) von Willebrand factor (VWF) on endothelial cells1 and soluble VWF in the flowing blood2;3 or at site of injury where VWF-rich platelet thrombi are formed4-6. This cleavage is highly specific occurring at the Tyr1605-Met1606 bond in the A2 domain7. In vivo, fluid shear stress accelerates the cleavage of cell bound ULVWF1;8 and soluble VWF in circulation2;3. In vitro addition of a denaturant such as urea9 or guanidine7 markedly accelerates the cleavage of soluble VWF by ADAMTS13. These findings facilitate the development of various biochemical assays for assessing ADAMTS13 activity.
The importance of VWF proteolysis is highlighted by the development of a fatal syndrome thrombotic thrombocytopenic purpura (TTP) when plasma ADAMTS13 activity is severely deficient, either due to hereditary mutations of ADAMTS13 gene10 or acquired formation of autoantibodies that inhibit ADAMTS13 activity11-13. Nearly all adult patients with severely deficient ADAMTS13 activity harbor polyclonal immunoglobulin Gs (IgGs) that bind the Cys-rich and spacer domains, particularly the spacer domain of ADAMTS1313-17. Recent studies have shown that exosite 3 (i.e. Y659-Y665) and several other adjacent amino acid residues (i.e. R568 and F592) in the spacer domain comprise a major antigenic epitope for autoantibodies in TTP18;19. This region is also found to play an essential role in proteolytic cleavage of VWF under various conditions6;20-24 and inhibition of arterial thrombus formation in vivo6.
Clearly a need exists for the identification of those residues in the ADAMTS13 spacer region which modulate substrate recognition and autoantibody recognition. This information should provide guidance for the development of therapeutic compositions useful for the treatment of TTP, myocardial infarction and stroke, and other inflammatory and arterial thrombotic disorders.